18/06/06

High-­‐Performance  Liquid  
Chromatography  

Characteris9cs  of  HPLC  

• Mostly  used  for  compounds  that  could  not  be  
vola9lized.  

• Generally  involves  adsorp9on  chromatography.  

• Requires  high  pressure  to  push  the  mobile  

phase  through  a  compact  column.  

Image  credit:  David  Harvey,  Chemistry  LibreTexts,  UC  Davis  

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 18/06/06

Why  High  Pressure  Gives  High  

Performance  
• The  efficiency  of  a  column  increases  as  the  
compactness  of  its  sta9onary  phase  increases.  

D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

Why  High  Pressure  Gives  High  

Performance  
• The  efficiency  of  a  column  increases  as  the  
compactness  of  its  sta9onary  phase  increases.  

• A  more  uniform  flow  can  reduce  the  mul9ple  

paths  term  

Redrawn  from:  D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

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 18/06/06

Why  High  Pressure  Gives  High  

Performance  
• The  efficiency  of  a  column  increases  as  the  
compactness  of  its  sta9onary  phase  increases.  

• A  more  uniform  flow  can  reduce  the  mul9ple  

paths  term  

• Less  diffusion  required  for  a  solute  to  reach  

the  sta9onary  phase  

Redrawn  from:  D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

Structure  of  an  HPLC  unit  

Gradient  valve  
Solvent  degasser   Mixing  vessel  

Pump  

Injec9on  valve  
Solvent  reservoirs  
(solvents  with  different  polarity)  

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Structure  of  an  HPLC  unit  

Inject  posi9on   Loading  posi9on  
Solvent  input   Solvent  input  

To  column  
To  column  

Waste   Waste  

Injec9on  loop   Sample  saved  in  

Injec9on   loop  
the  injec9on   loop  

Structure  of  an  HPLC  unit  

Gradient  valve  
Solvent  degasser   Mixing  vessel   Data  Processor  

Detector  

Pump  

Guard  column  
Analy9cal  Column  
Injec9on  valve   Waste/Frac9on  
Solvent  reservoirs   collector  
(solvents  with  different  polarity)  

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Types  of  HPLC  Columns

• Guard  column  (or  pre-­‐column)  
– To  protect  the  analy9cal  column  

• Analy9cal  column  
– To  separate  the  samples  

Image  credit:  Chemicals  Evalua9on  and  Research  Ins9tute,  Japan  

Types  of  HPLC  Columns

• Guard  column  (or  pre-­‐column)  
– To  protect  the  analy9cal  column  

Sample  injec9on  
 
 
– The  same  sta9onary  phase  as  the  analy9cal  column  

– Larger  par9cle  size  

Image  credit:  Chemicals  Evalua9on  and  Research  Ins9tute,  Japan  

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 18/06/06

Types  of  HPLC  Analy9cal  Columns

• Reversed-­‐phase  chromatography  
– Polar  mobile  phase;  non-­‐polar  sta9onary  phase  
– Most  common  mode  of  HPLC  

• Normal-­‐phase  chromatography  
– Non-­‐polar  mobile  phase;  polar  sta9onary  phase  

Types  of  HPLC  Analy9cal  Columns

• Reversed-­‐phase  chromatography  
– Polar  mobile  phase;  non-­‐polar  sta9onary  phase  
– Most  common  mode  of  HPLC  

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Isocra9c  and  Gradient  Elu9on  

• Isocra9c  elu9on  
– An  HPLC  run  with  constant  mobile  phase  

• Gradient  elu9on  
– An  HPLC  run  with  changing  mobile  phase  composi9on  

Isocra9c  and  Gradient  Elu9on  

• Isocra9c  elu9on  
– An  HPLC  run  with  constant  mobile  phase  
Reversed-­‐phase  liquid  chromatography  
Solvent  A:  Polar  solvent  
Solvent  B:  Non-­‐polar  solvent  

D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

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 18/06/06

Isocra9c  and  Gradient  Elu9on  

• Isocra9c  elu9on  
– An  HPLC  run  with  constant  mobile  phase  

• Gradient  elu9on  
– An  HPLC  run  with  changing  mobile  phase  composi9on  

Reversed  phase  liquid  chromatography  

Solvent  A:  Polar  solvent  
Solvent  B:  Non-­‐polar  solvent  

D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

Detectors  

Type of Detector   Applicable Samples   Detection Limit  

Mass Spectrometer (MS)   Tunable for any sample   ng order  

Spectrophotometry   Universal / selective   ng ~ pg  
Electrochemical   Redox-active compounds   fg ~ pg  
Chemiluminescence (CS)   Oxidative reagent   .002 to .02 µg/L  
Evaporative Light Scattering   Universal   High ng  
Charged Aerosol   Hydrocarbons   Low ng  

Table  modified  from:  D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

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Detectors  
• Evapora9ve  light   • Charged  aerosol  
sca]ering  detector   detector  

D.  Harris,  Quan9ta9ve  Chemical  Analysis,  9th  ed.  

Take-­‐home  Message  

High-­‐performance  liquid  chromatography  

outperforms  tradi9onal  liquid  chromatography  
due  to  their  compact  columns!